KR102463682B1 - Viscosity-reducing excipient compounds for protein formulations - Google Patents

Abstract

The present invention includes formulations allowing the delivery of concentrated protein solutions and methods for their production. The method of the present invention can yield lower viscosity liquid formulations or higher concentrations of therapeutic or non-therapeutic proteins in liquid formulations compared to traditional protein solutions.

Description

Viscosity-Reducing Excipient Compounds for Protein Formulations

Related applications

This application is a U.S. Provisional Application No. 62/014,784 filed on June 20, 2014, U.S. Provisional Application No. 62/083,623 filed on November 24, 2014, and a U.S. Provisional Application filed on March 23, 2015 Claims the benefit of Patent No. 62/136,763. The entirety of each of these applications is incorporated herein by reference.

technical field

This application relates generally to formulations for delivering biopolymers.

Biopolymers can be used for therapeutic or non-therapeutic purposes. Biopolymer-based therapies, such as antibody or enzyme formulations, are widely used to treat disease. Non-therapeutic biopolymers such as enzymes, peptides and structural proteins have utility in non-therapeutic applications such as household, nutritional, commercial and industrial uses.

Biopolymers used in therapeutic applications must be formulated to permit their introduction into the body for the treatment of disease. For example, instead of administering the formulations by intravenous (IV) injection, it is advantageous under certain circumstances to deliver antibody and protein/peptide biopolymer formulations by the subcutaneous (SC) or intramuscular (IM) route. To better improve patient compliance and comfort with SC or IM injection, the liquid volume in the syringe is typically limited to 2-3 cc, and the viscosity of the formulation is typically lower than about 20 centipoise (cP), but Thus, the formulation can be delivered using conventional medical devices and small caliber needles. These delivery parameters do not always match the dosage requirements for the formulation being delivered.

For example, antibodies may need to be delivered at high dose levels to exert their intended therapeutic effect. Using limited liquid volumes to deliver high dose levels of antibody formulations may require high concentrations of antibody in the delivery vehicle, sometimes exceeding the 150 mg/ml level. At this dosage level, the viscosity versus concentration plot of the protein solution lies outside their linear-non-linear conversion range, such that the viscosity of the formulation rises dramatically as the viscosity increases. However, the increased viscosity is not compatible with standard SC or IM delivery systems. Biopolymer-based therapeutic solutions are also prone to stability issues such as precipitation, hazing, opalescence, denaturation and gel formation, reversible or irreversible aggregation. Stability issues either limit the shelf life of the solution, or require special conditioning.

One approach to creating an injectable protein formulation is to convert a therapeutic protein solution into a powder that can be reconstituted to form a suspension suitable for SC or IM delivery. Lyophilization is a standard technique for producing protein powders. Lyophilization, spray drying and even supercritical fluid extraction after precipitation were used to generate protein powders for subsequent reconstitution. Powder suspensions are less viscous prior to redissolving (relative to solutions at the same total dose) and thus may be suitable for SC or IM injection, provided that the particles are small enough to pass through a needle. However, protein crystals present in the powder have an innate risk of triggering an immune response. Unspecified protein stability/activity after redissolving poses an additional problem. There remains a need in the art for techniques to create low viscosity protein formulations for therapeutic purposes, while avoiding the limitations introduced by protein powder suspensions.

In addition to the therapeutic uses of the proteins described above, biopolymers such as enzymes, peptides and structural proteins can be used for non-therapeutic uses. These non-therapeutic biopolymers can be produced from a number of different routes, for example, derived from plant sources, animal sources, or produced from cell cultures.

Non-therapeutic proteins can be produced, transported, stored and controlled, usually in water as granular or powdery materials or as solutions or dispersions. Biopolymers for non-therapeutic use may be granular or fibrous proteins, and certain forms of these materials may have limited solubility in water or may exhibit high viscosity upon dissolution. These solution properties can present challenges to the formulation, control, storage, pumping and performance of non-therapeutic substances, and therefore there is a need to reduce the viscosity, improve solubility and stability of non-therapeutic protein solutions. .

Proteins are complex biopolymers, each with a uniquely folded 3-D structure and surface energy map (hydrophobic/hydrophilic regions and charges). In concentrated protein solutions, these macromolecules can strongly interact and even interlock with each other in complex ways, depending on their precise shape and surface energy distribution. "Hot-spots" for strong specific interactions cause protein clustering, increasing solution viscosity. To address these issues, a number of excipient compounds are used in biotherapeutic formulations aimed at reducing solution viscosity by delaying local interactions and by clustering. These efforts are individually tailored and often guided by empirical and sometimes in silico simulations. Combinations of excipient compounds may be provided, but optimizing such combinations should also proceed empirically and on a case-by-case basis.

There remains a need in the art for a virtually universal approach to the reduced viscosity of protein formulations at a given concentration under non-linear conditions. There is a further need in the art to achieve this viscosity reduction while preserving the activity of the protein. It would further be desirable to adapt a viscosity-reducing system for use with formulations having a tunable and sustained release profile and for use with formulations suitable for depot injection.

In an embodiment, disclosed herein is a liquid formulation comprising a protein and an excipient compound selected from the group consisting of hindered amines, anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids and low molecular weight aliphatic polyacids, the excipients The compound is added in a viscosity-reducing amount. In an embodiment, the protein is a PEGylated protein and the excipient is a low molecular weight aliphatic polyacid. In an embodiment, the formulation is a pharmaceutical composition and the therapeutic formulation comprises a therapeutic protein, wherein the excipient compound is a pharmaceutically acceptable excipient compound. In an embodiment, the formulation is a non-therapeutic formulation and the non-therapeutic formulation comprises a non-therapeutic protein. In an embodiment, the viscosity-reducing amount reduces the viscosity of the formulation to a viscosity less than that of the control formulation. In an embodiment, the viscosity of the formulation is at least about 10% less than the viscosity of the control formulation, or at least about 30% less than the viscosity of the control formulation, or at least about 50% less than the viscosity of the control formulation, or at least about the viscosity of the control formulation. less than 70%, or at least about 90% less than the viscosity of the control formulation. In an embodiment, the viscosity is less than about 100 cP, or less than about 50 cP, or less than about 20 cP, or less than about 10 cP. In an embodiment, the excipient compound has a molecular weight of less than 5000 Da, or less than 1500 Da, or less than 500 Da. In an embodiment, the formulation contains at least about 25 mg/ml of protein, or at least about 100 mg/ml of protein, or at least about 200 mg/ml of protein, or at least about 300 mg/ml of protein. In an embodiment, the formulation comprises from about 5 mg/ml to about 300 mg/ml of the excipient compound, or from about 10 mg/ml to about 200 mg/ml of the excipient compound, or about 20 mg/ml to the excipient compound. to about 100 mg/ml, or from about 25 mg/ml to about 75 mg/ml of the excipient compound. In an embodiment, the formulation has improved stability when compared to a control formulation. In an embodiment, the excipient compound is a hindered amine. In an embodiment, the hindered amine is selected from the group consisting of caffeine, theophylline, tyramine, procaine, lidocaine, imidazole, aspartame, saccharin, and acesulfame potassium. In an embodiment, the hindered amine is caffeine. In an embodiment, the hindered amine is an anesthetic compound for local injection. The hindered amine may have independent pharmaceutical properties, and the hindered amine may be present in the formulation in an amount having an independent pharmaceutical effect. In embodiments, the hindered amine may be present in the formulation in an amount less than a therapeutically effective amount. The independent pharmaceutical activity may be local anesthetic activity. In an embodiment, a hindered amine having independent pharmaceutical activity is combined with a second excipient compound that further reduces the viscosity of the formulation. The second excipient compound may be selected from the group consisting of caffeine, theophylline, tyramine, procaine, lidocaine, imidazole, aspartame, saccharin and acesulfame potassium. In embodiments, the formulation may include additional agents selected from the group consisting of preservatives, surfactants, sugars, polysaccharides, arginine, proline, hyaluronidase, stabilizers, and buffers.

Disclosed herein is a method of treating a disease or disorder in a mammal comprising administering to the mammal a liquid therapeutic formulation, wherein the therapeutic formulation comprises a therapeutically effective amount of a therapeutic protein, wherein the formulation comprises a sterically hindered formulation. further comprising a pharmaceutically acceptable excipient compound selected from the group consisting of amines, anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids, and low molecular weight aliphatic polyacids; The therapeutic formulation is effective for the treatment of a disease or disorder. In an embodiment, the therapeutic protein is a pegylated protein and the excipient compound is a low molecular weight aliphatic polyacid. In an embodiment, the excipient is a hindered amine. In an embodiment, the hindered amine is a local anesthetic compound. In an embodiment, the formulation is administered by subcutaneous injection, or intramuscular injection, or intravenous injection. In an embodiment, the excipient compound is present in the therapeutic formulation in a viscosity-reducing amount, wherein the viscosity-reducing amount reduces the viscosity of the therapeutic formulation to a viscosity less than that of the control formulation. In an embodiment, the therapeutic formulation has improved stability as compared to a control formulation. In an embodiment, the excipient compound is essentially pure.

Further disclosed herein is a method of reducing pain at the site of injection of a therapeutic protein in a mammal in need thereof comprising administering a liquid therapeutic formulation by injection, wherein the treatment The therapeutic formulation comprises a therapeutically effective amount of a therapeutic protein, the formulation further comprises a pharmaceutically acceptable excipient compound selected from the group consisting of an anesthetic compound for local injection, wherein the pharmaceutically acceptable excipient compound comprises a viscosity-reducing amount added to the formulation; Mammals experience less pain by administration of a therapeutic formulation comprising an excipient compound than by administration of a control therapeutic formulation, and the control therapeutic formulation does not contain an excipient compound, and is otherwise combined with the therapeutic formulation. same.

As used herein, in embodiments, a liquid protein formulation comprising a therapeutic protein and an excipient compound selected from the group consisting of hindered amines, anionic aromatics, functionalized amino acids, oligopeptides and short chain organic acids and low molecular weight aliphatic polyacids is prepared Disclosed is a method of improving the stability of a liquid protein formulation comprising the steps of: wherein the liquid protein formulation demonstrates improved stability as compared to a control liquid protein formulation, wherein the control liquid protein formulation does not contain an excipient compound; Same as liquid protein formulation. The stability of the liquid formulation may be cold storage condition stability, room temperature stability or elevated temperature stability.

Also in an embodiment, disclosed herein is a liquid formulation comprising a protein and an excipient selected from the group consisting of hindered amines, anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids, and low molecular weight aliphatic polyacids, The presence of the excipient compound in the formulation results in improved protein-protein interaction characteristics as measured by the protein diffusion interaction parameter kD, or the second virial coefficient B22. In an embodiment, the formulation is a therapeutic formulation and comprises a therapeutic protein. In an embodiment, the formulation is a non-therapeutic formulation and comprises a non-therapeutic protein.

Further, in an embodiment, disclosed herein is a method of improving a protein-related process comprising providing the liquid formulation described above and using it in a processing method. In an embodiment, the processing method comprises filtration, pumping, mixing, centrifugation, membrane separation, lyophilization or chromatography.

Disclosed herein are formulations and methods of producing formulations that allow for the delivery of concentrated protein solutions. In embodiments, the approaches disclosed herein may yield lower viscous liquid formulations or higher concentrations of therapeutic or non-therapeutic proteins in liquid formulations compared to traditional protein solutions. In embodiments, the approaches disclosed herein can yield liquid formulations with improved stability when compared to traditional protein solutions. A stable formulation retains its physical and chemical stability in which the protein contained therein retains its therapeutic or non-therapeutic efficacy upon storage under storage conditions, whether cold storage conditions, room temperature conditions or elevated temperature storage conditions. Advantageously, stable formulations may also provide protection against aggregation or precipitation of proteins dissolved therein. For example, cold storage conditions may entail storage in a refrigerator or freezer. In some examples, cold storage conditions may involve conventional refrigerator or freezer storage at a temperature of 10° C. or less. In a further example, cold storage conditions involve storage at a temperature of about 2°C to about 10°C. In another example, cold storage conditions involve storage at a temperature of about 4°C. In a further example, cold storage conditions involve storage at a freezing temperature, such as about 0° C. or less. In another example, cold storage conditions involve storage at a temperature of about -30°C to about 0°C. Room temperature storage conditions may entail storage at ambient temperature, for example, from about 10°C to about 30°C. For example, elevated temperature stability at temperatures from about 30°C to about 50°C can be used as part of an accelerated aging study to predict long-term storage at typical ambient (10-30°C) conditions.

It is well known to those skilled in the art of polymer science and engineering that proteins in solution tend to form entanglements, which can limit the translational mobility of entangled chains and interfere with the therapeutic or non-therapeutic efficacy of proteins. In an embodiment, an excipient compound as disclosed herein is capable of inhibiting protein clustering due to a specific interaction between the excipient compound and a target protein in solution. Excipient compounds as disclosed herein may be natural or synthetic, and are preferably substances generally recognized by the FDA as safe (“GRAS”).

1. Definition

For the purposes of this disclosure, the term “protein” refers to an amino acid sequence having a chain long enough to produce a separate tertiary structure, typically having a molecular weight of 1 to 3000 kD. In some embodiments, the molecular weight of the protein is between about 50 and 200 kD; In other embodiments, the molecular weight of the protein is between about 20 and 1000 kD or between about 20 and 2000 kD. In contrast to the term “protein”, the term “peptide” refers to an amino acid sequence that does not have a distinct tertiary structure. A wide variety of biopolymers are included within the scope of the term “protein”. For example, the term “protein” may refer to therapeutic or non-therapeutic proteins, including antibodies, aptamers, fusion proteins, pegylated proteins, synthetic polypeptides, protein fragments, lipoproteins, enzymes, structural peptides, and the like. can

By way of non-limiting example, therapeutic proteins include mammalian proteins such as hormones and prohormones (eg, insulin and proinsulin, glucagon, calcitonin, thyroid hormone (T3 or T4 or thyroid stimulating hormone), parathyroid hormone, follicle stimulating hormone). , luteinizing hormone, growth hormone, growth hormone releasing factor, etc.); coagulation and anticoagulant factors (eg, tissue factor, von Willebrand factor, factor VIIIC, factor IX, protein C, plasminogen activator (urokinase, tissue type plasminogen activator), thrombin); cytokines, chemokines and inflammatory mediators; interferon; colony-stimulating factors; interleukins (eg, IL-1 to IL-10); growth factors (eg, vascular endothelial growth factor, fibroblast growth factor, platelet-derived growth factor, transforming growth factor, neurotrophic growth factor, insulin-like growth factor, etc.); albumin; collagen and elastin; hematopoietic factors (eg, erythropoietin, thrombopoietin, etc.); osteoinductive factors (eg, bone morphogenetic proteins); receptors (eg, integrins, cadherins, etc.); surface membrane proteins; transport protein; regulatory proteins; antigenic proteins (eg, viral components that act as antigens); and antibodies. The term "antibody" herein, in its broadest scope, includes, by way of non-limiting example, monoclonal antibodies (including, for example, full-length antibodies having an immunoglobulin Fc region), single chain molecules, bispecific and multispecific antibodies, dire body, antibody compositions having polyepitopic specificity, and antibody fragments (including, for example, Fab, Fv, and F(ab')2). Antibodies may also be referred to as “immunoglobulins”. Antibodies are understood to relate to specific protein or non-protein “antigens” that are biologically important substances; Administration of a therapeutically effective amount of an antibody to a patient may alter its biological properties by complexing with the antigen, and thus the patient experiences a therapeutic effect.

In an embodiment, the proteins are pegylated, meaning that they comprise poly(ethylene glycol) (“PEG”) and/or poly(propylene glycol) (“PPG”) units. PEGylated proteins or PEG-protein conjugates have found utility in therapeutic applications due to their advantageous properties such as solubility, pharmacokinetics, pharmacodynamics, immunogenicity, renal clearance and stability. Non-limiting examples of pegylated proteins include pegylated interferon (PEG-IFN), pegylated anti-VEGF, PEG protein conjugate drug, adagen, pegasparagase, pegfilgrastim, pegloticase. , pegvisomant, pegylated epoetin-β, and sertorizumab pegol.

PEGylated proteins can be synthesized by a variety of methods, such as reaction of a protein with a PEG reagent having one or more reactive functional groups. Reactive functional groups for PEG reagents are capable of forming bonds with proteins at targeted protein sites, such as lysine, histidine, cysteine and the N-terminus. Typical pegylation reagents have reactive functional groups such as aldehyde, maleimide, or succinimide groups that have specific reactivity with amino acid residues targeted to the protein. The PEGylated reagent may have a PEG chain of from about 1 to about 1000 PEG and/or PPG repeat units. Another method of pegylation involves glycopegylation, wherein the protein is first glycosylated, and then the glycosylated residues are pegylated in a second step. Certain pegylation processes are assisted by enzymes such as sialyltransferase and transglutaminase.

While pegylated proteins may provide therapeutic advantages over native, non-pegylated proteins, these materials may have physical or chemical properties that make them difficult to purify, dissolve, filter, concentrate and administer. Pegylation of proteins can result in higher solution viscosities compared to native proteins, which generally require formulation of pegylated protein solutions at lower concentrations.

It may be desirable to formulate protein therapeutics in stable, low viscosity solutions so that they can be administered to patients in minimally injectable solutions. For example, subcutaneous (SC) or intramuscular (IM) injections of drugs generally require small injection volumes, preferably less than 2 ml. The SC and IM injection routes are suitable for self-administering treatment, which is a less expensive and more accessible form of intravenous (IV) injection, which is performed only under direct medical supervision. Formulations for SC or IM injection require low solution viscosities, generally less than 30 cP, and preferably less than 20 cP, to allow easy flow of the treatment solution through a narrow gauge needle. This combination of small injection volumes and low viscosity requirements presents a challenge for the use of pegylated protein therapy in the SC or IM injection route.

A protein of interest having a therapeutic effect may be referred to as a “therapeutic protein”; A formulation containing a therapeutic protein in a therapeutically effective amount may be referred to as a “therapeutic formulation”. A therapeutic protein contained in a therapeutic formulation may also be referred to as its “protein active ingredient”. Typically, therapeutic formulations comprise a therapeutically effective amount of a protein active ingredient and excipients, with or without other optional ingredients. As used herein, the term “therapeutic” includes both treatment of an existing disorder and prevention of the disorder.

"Treatment" includes any measure intended to manage, cure, alleviate, ameliorate, treat or otherwise beneficially affect a disorder, including preventing or delaying the onset of symptoms and/or alleviating or ameliorating the symptoms of the disorder. . Those patients in need of treatment include both those already with the particular disorder and those for whom prevention of the disorder is desired. A disorder is any condition that alters the homeostatic well-being of a mammal, including an acute or chronic disease, or a pathological condition that predisposes the mammal to an acute or chronic disease. Non-limiting examples of disorders include cancer, metabolic disorders (eg diabetes), allergic disorders (eg asthma), skin disorders, cardiovascular disorders, respiratory disorders, hematological disorders, musculoskeletal disorders, inflammatory or rheumatic disorders, autologous immune disorders, gastrointestinal disorders, urinary disorders, reproductive disorders, neurological disorders, and the like. For therapeutic purposes, the term “mammal” may refer to any animal classified as a mammal, including humans, domestic animals, pets, farm animals, sports animals, work animals, and the like. Thus, “treatment” may include both veterinary and human treatment. For convenience, a mammal receiving such “treatment” may be referred to as a “patient”. In certain embodiments, the patient can be of any age, including an intrauterine fetal animal.

In an embodiment, treatment involves providing a therapeutically effective amount of a therapeutic formulation to a mammal in need thereof. A "therapeutically effective amount" is at least a minimal concentration of a therapeutic protein administered to a mammal in need thereof to achieve treatment of an existing disorder or prevention of the foreseen disorder (such treatment or prevention is a "therapeutic intervention"). . Therapeutically effective amounts of various therapeutic proteins that may be included as active ingredients in therapeutic formulations may be familiar in the art; Or for a therapeutic protein that is later discovered or applied for therapeutic intervention herein, a therapeutically effective amount can be determined by standard techniques performed by one of ordinary skill in the art using no more than routine experimentation.

A protein in question used for a non-therapeutic purpose (ie, a purpose not involving treatment), such as household, nutritional, commercial and industrial uses, may be referred to as a “non-therapeutic protein”. Formulations containing non-therapeutic proteins may be referred to as “non-therapeutic formulations”. Non-therapeutic proteins may be derived from plant sources, animal sources, or produced from cell culture; They may also be enzymes or structural proteins. Non-therapeutic proteins can be used in household, nutritional, commercial and industrial applications such as catalysts, human and animal nutrition, processing aids, detergents and waste treatment.

An important category of non-therapeutic biopolymers are enzymes. Enzymes have many non-therapeutic uses, such as catalysts, human and animal nutritional components, processing aids, detergents and waste disposal agents. Enzyme catalysts are used to accelerate various chemical reactions. Examples of enzyme catalysts for non-therapeutic use include catalase, oxidoreductase, transferase, hydrolase, lyase, isomerase and ligase. Human and animal nutritional uses of enzymes include nutraceuticals, nutritional sources of proteins, chelation or controlled delivery of micronutrients, digestive aids and supplements; They may be derived from amylases, proteases, trypsin, lactases, and the like. Enzyme processing aids are useful for improving the production of food and beverage products in operations such as baking, brewing, fermentation, juice processing and winemaking. Examples of these food and beverage processing aids include amylase, cellulase, pectinase, glucanase, lipase and lactase. Enzymes can also be used in the production of biofuels. Ethanol for biofuels can be assisted, for example, by enzymatic digestion of biomass feedstocks such as cellulosic and lignocellulosic materials. Treatment of these feedstocks with cellulose and ligninase converts the biomass into a substrate that can be fermented as a fuel. In commercial applications, enzymes are used as detergents, detergents and dye lifting aids for laundry, dishwashing, surface cleaning and tool cleaning applications. Typical enzymes for this purpose include proteases, celluloses, amylases and lipases. Additionally, non-therapeutic enzymes are used in a variety of commercial and industrial processes, such as textile softening with cellulase, leather processing, waste treatment, soil sediment treatment, water treatment, pulp bleaching and pulp softening and debonding. Typical enzymes for these purposes are amylases, xylanases, cellulases and ligninases.

Other examples of non-therapeutic biopolymers include fibrous or structural proteins such as keratin, collagen, gelatin, elastin, fibroin, actin, tubulin or hydrolyzed, degraded or derivatized forms thereof. These substances are used in the manufacture and formulation of food ingredients such as gelatin, ice cream, yogurt and confectionery; They are also added to foods as thickeners, flow modifiers, mouth feel improvers and as a source of nutritional protein. In the cosmetic and personal care industry, collagen, elastin, keratin and hydrolyzed keratin are widely used as ingredients in skin care and hair care formulations. Another example of a non-therapeutic biopolymer is whey protein, such as beta-lactoglobulin, alpha-lactabumin, and serum albumin. These whey proteins are produced on a large scale as a by-product from dairy operations and have been used for a variety of non-therapeutic uses.

2. Therapeutic formulation

In one aspect, the formulations and methods disclosed herein provide a stable liquid formulation of improved or reduced viscosity comprising a therapeutic protein and an excipient compound in a therapeutically effective amount. In embodiments, the formulation may provide an acceptable concentration of active ingredient and an acceptable viscosity while improving stability. In an embodiment, the formulation provides an improvement in stability as compared to a control formulation; For the purposes of the present disclosure, a control formulation is a formulation containing the protein active ingredient of the same dry weight in all methods for a therapeutic formulation, except that there is no excipient compound. In an embodiment, the improved stability of the protein containing formulation is a lower percentage of soluble aggregates, particulates, microscopically visible particles or gel-forming forms compared to the control formulation.

The viscosity of a liquid protein formulation depends on the nature of the protein itself (eg, enzyme, antibody, receptor, fusion protein, etc.); its size, three-dimensional structure, chemical composition and molecular weight; its concentration in the formulation; components of the formulation other than protein; the desired pH range; storage conditions for the formulation; and how the formulation is administered to a patient. Therapeutic proteins most suitable for use with the excipient compounds described herein are preferably essentially pure, ie, free of contaminating proteins. In an embodiment, an "essentially pure" therapeutic protein is a protein composition comprising at least 90%, preferably at least 95%, or more preferably, at least 99%, by weight of the therapeutic protein, all of the composition It is based on the total weight of the therapeutic protein and the contaminating protein in the body. For purposes of clarity, proteins added as excipients are not intended to be included in this definition. The therapeutic formulations described herein are pharmaceutical grade formulations, i.e. mammals, in such a form that the desired therapeutic efficacy of the protein active ingredient can be achieved and without containing ingredients that are toxic to the mammal to which the formulation is administered. It is intended for use as a formulation intended for use in treating

In an embodiment, the therapeutic formulation contains at least 25 mg/ml of protein active ingredient. In another embodiment, the therapeutic formulation contains at least 100 mg/ml of protein active ingredient. In another embodiment, the therapeutic formulation contains at least 200 mg/ml of protein active ingredient. In another embodiment, the therapeutic formulation solution contains at least 300 mg/ml of protein active ingredient. Generally, the excipient compounds disclosed herein are added to the therapeutic formulation in an amount from about 5 to about 300 mg/ml. In an embodiment, the excipient compound may be added in an amount from about 10 to about 200 mg/ml. In an embodiment, the excipient compound may be added in an amount from about 20 to about 100 mg/ml. In an embodiment, the excipient may be added in an amount of about 25 to about 75 mg/ml.

Excipient compounds of various molecular weights are selected for certain advantageous properties when combined with the protein active ingredient in the formulation. Examples of therapeutic formulations comprising an excipient compound are provided below. In an embodiment, the excipient compound has a molecular weight of less than 5000 Da. In an embodiment, the excipient compound has a molecular weight of less than 1000 Da. In an embodiment, the excipient compound has a molecular weight of less than 500 Da.

In an embodiment, an excipient compound disclosed herein is added to the therapeutic formulation in a viscosity-reducing amount. In an embodiment, the viscosity-reducing amount is an amount of an excipient compound that reduces the viscosity of the formulation by at least 10% as compared to a control formulation; For the purposes of the present disclosure, a control formulation is a formulation containing the protein active ingredient of the same dry weight in all methods for a therapeutic formulation, except that there is no excipient compound. In an embodiment, the viscosity-reducing amount is an amount of an excipient compound that reduces the viscosity of the formulation by at least 30% as compared to a control formulation. In an embodiment, the viscosity-reducing amount is an amount of an excipient compound that reduces the viscosity of the formulation by at least 50% as compared to a control formulation. In an embodiment, the viscosity-reducing amount is an amount of an excipient compound that reduces the viscosity of the formulation by at least 70% as compared to a control formulation. In an embodiment, the viscosity-reducing amount is an amount of an excipient compound that reduces the viscosity of the formulation by at least 90% as compared to a control formulation.

In an embodiment, the viscosity-reducing amount yields a therapeutic formulation having a viscosity of less than 100 cP. In another embodiment, the therapeutic formulation has a viscosity of less than 50 cP. In another embodiment, the therapeutic formulation has a viscosity of less than 20 cP. In another embodiment, the therapeutic formulation has a viscosity of less than 10 cP. As used herein, the term “viscosity” refers to a value of mechanical viscosity as measured by the methods disclosed herein.

Therapeutic formulations according to the present disclosure have certain advantageous properties. In embodiments, the therapeutic formulation is resistant to shear degradation, phase separation, turbidity, precipitation and denaturation. In embodiments, the therapeutic formulation is processed, purified, stored, infused, administered, filtered and centrifuged more effectively than a control formulation. In an embodiment, the therapeutic formulation is administered to the patient at a high concentration of the therapeutic protein. In an embodiment, the therapeutic formulation is administered to the patient with less discomfort than would be experienced by a similar formulation without the therapeutic excipient. In an embodiment, the therapeutic formulation is administered as a depot injection. In an embodiment, the therapeutic formulation prolongs the half-life of the therapeutic protein in the body. These characteristics of the therapeutic formulations as disclosed herein are characteristic of the clinical situation, ie, a small bore needle typical for IM/SC purposes and tolerable (eg 2-3 cc) injection volumes for patient acceptance of intramuscular injection. administration of such formulations by intramuscular or subcutaneous injection in cases involving the use of In contrast, injections of similar doses of therapeutic protein using conventional formulation techniques are limited by the higher viscosity of conventional formulations, and thus SC/IM injections of conventional formulations are not suitable for clinical situations.

In an embodiment, the therapeutic excipient has antioxidant properties that stabilize the therapeutic protein against oxidative damage. In an embodiment, the therapeutic formulation is stored for extended periods of time at ambient temperature or in refrigerated conditions without significant loss of efficacy of the therapeutic protein. In an embodiment, the therapeutic formulation is dried for storage until it is needed; It is then reconstituted using a suitable solvent, for example water. Advantageously, formulations as described herein may be stable over long periods of time from months to years. When exceptionally long storage is desired, the formulation can be stored in the freezer (and subsequently reactivated) without fear of protein denaturation. In embodiments, the formulation may be prepared for long-term storage that does not require refrigeration.

Methods for preparing therapeutic formulations may be familiar to those skilled in the art. Therapeutic formulations of the invention can be prepared, for example, by adding an excipient compound to the formulation before or after the therapeutic protein is added to the solution. A therapeutic formulation can be produced, for example, by combining a therapeutic protein with an excipient at a first (lower) concentration followed by filtration or centrifugation to produce a second (higher) therapeutic protein. can be processed. Therapeutic formulations may consist of chaotropes, cosmotropes, hydrotropes and salts and one or more excipient compounds. Therapeutic formulations may be comprised of one or more excipient compounds using techniques such as encapsulation, dispersion, liposome, vesicular formulation, and the like. Methods for preparing a therapeutic formulation comprising an excipient compound disclosed herein may include a combination of excipient compounds. In embodiments, the combination of excipients may produce an advantage in lower viscosity, improved stability, or reduced injection site pain. Other additives may be incorporated into the therapeutic formulation during their manufacture including preservatives, surfactants, sugars, sucrose, trehalose, polysaccharides, arginine, proline, hyaluronidase, stabilizers, buffers, and the like. As used herein, pharmaceutically acceptable excipient compounds are those that are non-toxic and suitable for animal and/or human administration.

3. Non-therapeutic formulations

In one aspect, the formulations and methods disclosed herein provide a stable liquid formulation of improved or reduced viscosity comprising an effective amount of a non-therapeutic protein and an excipient compound. In an embodiment, the formulation improves stability, while providing an acceptable concentration of active ingredient and an acceptable viscosity. In an embodiment, the formulation provides an improvement in stability as compared to a control formulation; For the purposes of this disclosure, control formulations are formulations containing protein active ingredients that are the same dry weight in all ways relative to non-therapeutic formulations, except for the absence of excipient compounds.

The viscosity of a liquid protein formulation depends on the nature of the protein itself (eg, enzymes, structural proteins, degree of hydrolysis, etc.); its size, three-dimensional structure, chemical composition and molecular weight; its concentration in the formulation; formulation components other than proteins; the desired pH range; and storage conditions for the formulation.

In an embodiment, the non-therapeutic formulation contains at least 25 mg/ml of protein active ingredient. In another embodiment, the non-therapeutic formulation contains at least 100 mg/ml of protein active ingredient. In another embodiment, the non-therapeutic formulation contains at least 200 mg/ml of protein active ingredient. In another embodiment, the non-therapeutic formulation solution contains at least 300 mg/ml of protein active ingredient. Generally, the excipient compounds disclosed herein are added to the non-therapeutic formulation in an amount from about 5 to about 300 mg/ml. In an embodiment, the excipient compound is added in an amount from about 10 to about 200 mg/ml. In an embodiment, the excipient compound is added in an amount from about 20 to about 100 mg/ml. In an embodiment, the excipient is added in an amount of about 25 to about 75 mg/ml.

Excipient compounds of various molecular weights are selected for certain advantageous properties when combined with the protein active ingredient in the formulation. Examples of non-therapeutic formulations comprising excipient compounds are provided below. In an embodiment, the excipient compound has a molecular weight of less than 5000 Da. In an embodiment, the excipient compound has a molecular weight of less than 1000 Da. In an embodiment, the excipient compound has a molecular weight of less than 500 Da.

In an embodiment, an excipient compound disclosed herein is added to a non-therapeutic formulation in a viscosity-reducing amount. In an embodiment, the viscosity-reducing amount is an amount of an excipient compound that reduces the viscosity of the formulation by at least 10% as compared to a control formulation; For the purposes of the present disclosure, a control formulation is a formulation containing the protein active ingredient of the same dry weight in all methods for a therapeutic formulation, except that there is no excipient compound. In an embodiment, the viscosity-reducing amount is an amount of an excipient compound that reduces the viscosity of the formulation by at least 30% as compared to a control formulation. In an embodiment, the viscosity-reducing amount is an amount of an excipient compound that reduces the viscosity of the formulation by at least 50% as compared to a control formulation. In an embodiment, the viscosity-reducing amount is an amount of an excipient compound that reduces the viscosity of the formulation by at least 70% as compared to a control formulation. In an embodiment, the viscosity-reducing amount is an amount of an excipient compound that reduces the viscosity of the formulation by at least 90% as compared to a control formulation.

In an embodiment, the viscosity-reducing amount yields a non-therapeutic formulation having a viscosity of less than 100 cP. In another embodiment, the non-therapeutic formulation has a viscosity of less than 50 cP. In another embodiment, the non-therapeutic formulation has a viscosity of less than 20 cP. In another embodiment, the non-therapeutic formulation has a viscosity of less than 10 cP. As used herein, the term “viscosity” refers to a mechanical viscosity value.

Non-therapeutic formulations according to the present disclosure may have certain advantageous properties. In embodiments, the non-therapeutic formulation is resistant to shear degradation, phase separation, turbidity, precipitation, and denaturation. In embodiments, therapeutic formulations can be processed, purified, stored, pumped, filtered, and centrifuged more effectively than control formulations.

In an embodiment, the non-therapeutic excipient has antioxidant properties that stabilize the non-therapeutic protein against oxidative damage. In an embodiment, the non-therapeutic formulation is stored for extended periods of time at ambient temperature or in refrigerated conditions without significant loss of potency of the non-therapeutic protein. In an embodiment, the non-therapeutic formulation is dried for storage until it is needed; It can then be reconstituted using a suitable solvent, for example water. Advantageously, formulations as described herein are stable over long periods of time of months to years. When exceptionally long storage is desired, the formulation is preserved in the freezer (and subsequently reactivated) without fear of protein denaturation. In an embodiment, the formulation is prepared for long-term storage that does not require refrigeration.

Methods for preparing non-therapeutic formulations comprising the excipient compounds disclosed herein may be familiar to those skilled in the art. For example, the excipient compound may be added to the formulation before or after the non-therapeutic protein is added to the solution. The non-therapeutic formulation may be produced at a first (lower) concentration and then processed by filtration or centrifugation to produce a second (higher) concentration. Non-therapeutic formulations may consist of chaotropes, cosmotropes, hydrotropes and salts and one or more excipient compounds. Non-therapeutic formulations may consist of one or more excipient compounds using techniques such as encapsulation, dispersion, liposome, vesicular formulation, and the like. Other additives may be incorporated during their manufacture into non-therapeutic formulations including preservatives, surfactants, stabilizers, and the like.

4. Excipient Compounds

Several excipient compounds are described, each suitable for use with one or more therapeutic or non-therapeutic proteins, and allowing formulations to be formulated to contain protein(s) in high concentrations, respectively. Some categories of excipient compounds described below include: (1) hindered amines; (2) anionic aromatics; (3) functionalized amino acids; and (4) oligopeptides. Without wishing to be bound by theory, it is believed that the excipient compounds described herein relate to specific fragments, sequences, structures or segments of therapeutic proteins that are otherwise involved in interparticle (ie, protein-protein) interactions. The binding of these excipient compounds to therapeutic or non-therapeutic proteins can mask protein-protein interactions so that the protein can be formulated at high concentrations without causing excessive solution viscosity. The excipient compounds may advantageously be water-soluble and are therefore suitable for use with aqueous vehicles. In an embodiment, the excipient compound has a water solubility greater than 10 mg/ml. In an embodiment, the excipient compound has a water solubility greater than 100 mg/ml. In an embodiment, the excipient compound has a water solubility greater than 500 mg/ml. Advantageously for therapeutic proteins, the excipient compounds may be derived from biologically acceptable and non-immunogenic substances and are therefore suitable for pharmaceutical use. In therapeutic embodiments, the excipient compound may be metabolized within the body to yield a biologically compatible and non-immunogenic by-product.

a. Excipient compound category 1: hindered amines

High concentration solutions of therapeutic or non-therapeutic proteins can be formulated with small hindered amine molecules as excipient compounds. As used herein, the term “hindered amine” refers to a small molecule containing at least one bulky or hindered group consistent with the examples below. The hindered amines can be used in their free base form, in protonated form, or a combination of the two. In the protonated form, the hindered amine may be associated with an anionic counterion such as chloride, hydroxide, bromide, iodide, fluoride, acetate, formate, phosphate, sulfate or carboxylate. Hindered amine compounds useful as excipient compounds include secondary amines, tertiary amines, quaternary ammonium, pyridinium, pyrrolidone, pyrrolidine, piperidine, It may contain morpholine or guanidinium groups. The hindered amine compound also contains at least one bulky or hindered group, such as a cyclic aromatic, cycloaliphatic, cyclohexyl or alkyl group. In embodiments, the hindered group may itself be an amine group, such as a dialkylamine, trialkylamine, guanidinium, pyridinium or quaternary ammonium group. Without wishing to be bound by theory, it is believed that hindered amine compounds are associated with the aromatic moieties of proteins such as phenylalanine, tryptophan and tyrosine by cationic pi interactions. In embodiments, the cationic groups of hindered amines may have affinity for the electron-rich pi structure of aromatic amino acid residues in the protein, so that they shield these segments of the protein, thereby binding and aggregating these shielded amines. It can reduce the tendency of proteins.

In an embodiment, the hindered amine excipient compound is an imidazole, imidazoline, or imidazolidine group or a salt thereof, such as imidazole, 1-methylimidazole, 4-methylimidazole, 1-hexyl-3 -Methylimidazolium chloride, histamine, 4-methylhistamine, alpha-methylhistamine, betahistine, beta-alanine, 2-methyl-2-imidazoline, 1-butyl-3-methylimidazolium chloride, uric acid , potassium urate, betaazole, carnosine, aspartame, saccharin, acesulfame potassium, xanthine, theophylline, theobromine, caffeine and anserine. In an embodiment, the hindered amine excipient compound is dimethylethanolamine, dimethylaminopropylamine, triethanolamine, dimethylbenzylamine, dimethylcyclohexylamine, diethylcyclohexylamine, dicyclohexylmethylamine, hexamethylene Biguanide, poly(hexamethylene biguanide), imidazole, dimethylglycine, agmantine, diazabicyclo[2.2.2]octane, tetramethylethylenediamine, N,N-dimethylethanolamine, ethanolamine phosphate , Glucosamine, choline chloride, phosphocholine, niacinamide, isonicotinamide, N,N-diethyl nicotinamide, nicotinic acid sodium salt, tyramine, 3-aminopyridine, 2,4,6-trimethylpyridine, 3-pyridine Methanol, nicotinamide adenosine dinucrelotide, biotin, morpholine, N-methylpyrrolidone, 2-pyrrolidinone, procaine, lidocaine, dicyandiamide-taurine adduct, 2-pyridylethylamine, dicyandiamide-benzyl amine adduct, dicyandiamide-alkylamine adduct, dicyandiamide-cycloalkylamine adduct and dicyandiamide-aminomethanephosphonic acid adduct. In an embodiment, hindered amine compounds consistent with the present disclosure are formulated as protonated ammonium salts. In an embodiment, a hindered amine compound consistent with the present disclosure is formulated as a salt with an inorganic anion or an organic anion as the counterion. In an embodiment, a high concentration solution of a therapeutic or non-therapeutic protein is formulated with a combination of caffeine and benzoic acid, hydroxybenzoic acid or benzenesulfonic acid as an excipient compound. In an embodiment, the hindered amine excipient compound is metabolized in the body to yield a biologically compatible byproduct. In some embodiments, the hindered amine excipient compound is present in the formulation at a concentration of about 250 mg/ml or less. In a further embodiment, the hindered amine excipient compound is present in the formulation at a concentration of about 10 mg/ml to about 200 mg/ml. In yet a further aspect, the hindered amine excipient compound is present in the formulation at a concentration of about 20 to about 120 mg/ml.

In embodiments, certain hindered amine excipient compounds may have other pharmaceutical properties. As an example, xanthine is a category of hindered amines with independent pharmaceutical properties, including stimulant properties and bronchodilator properties when absorbed systemically. Representative xanthines include caffeine, aminophylline, 3-isobutyl-1-methylxanthine, paraxanthine, pentoxifylline, theobromine, theophylline, and the like. Methylated xanthines are understood to affect contractility, heart rate and bronchodilation. In some embodiments, the xanthine excipient compound is present in the formulation at a concentration of about 30 mg/ml or less.

Another category of hindered amines with independent pharmaceutical properties are local injectable anesthetic compounds. Local injectable anesthetic compounds are hindered amines having a three-component molecular structure of (a) a lipophilic aromatic ring, (b) an intermediate ester or amide linkage, and (c) a secondary or tertiary amine. This category of hindered amines is understood to interfere with nerve conduction by inhibiting the influx of sodium ions, thereby inducing local anesthesia. The lipophilic aromatic ring for a local anesthetic compound may consist of carbon atoms (eg, a benzene ring) or may contain heteroatoms (eg, a thiophene ring). Representative local injectable anesthetic compounds include amylocaine, articaine, bupivicaine, butacaine, butanylcaine, chlorprocaine, cocaine, cyclomethicaine, dimethocaine, editokaine, hexylcaine, isobucaine, levobupivacaine, lidocaine, metabutetamine, metabutoxycaine, mepivacaine, mepricaine, propoxycaine, prilocaine, procaine, piperocaine, tetracaine, trimecaine, etc. It is not limited to these. Anesthetic compounds for local injection may have a number of advantages in protein therapeutic formulations, such as reduced viscosity, improved stability, and reduced pain upon injection. In some embodiments, the local anesthetic compound is present in the formulation at a concentration of about 50 mg/ml or less.

In embodiments, hindered amines with independent pharmaceutical properties are used as excipient compounds according to the formulations and methods described herein. In some embodiments, an excipient compound with independent pharmaceutical properties does not have a pharmacological effect and/or is present in an amount that is not therapeutically effective. In another embodiment, an excipient compound with independent pharmaceutical properties has a pharmacological effect and/or is present in a therapeutically effective amount. In certain embodiments, hindered amines with independent pharmaceutical properties are used in combination with other excipient compounds selected to reduce formulation viscosity, wherein hindered amines with independent pharmaceutical properties benefit from their pharmaceutical activity. used to give For example, local injectable anesthetic compounds can be used to reduce formulation viscosity and also to reduce pain upon injection of the formulation. Reduction of injection pain may be caused by anesthetic properties; A lower injection force may also be required when the viscosity is reduced by the excipient. Alternatively, local injectable anesthetic compounds may be combined with other excipient compounds that reduce the viscosity of the formulation while conferring the desired pharmaceutical benefit of reduced local sensation during formulation injection.

b. Excipient Compound Category 2: Anionic Aromatics

High concentration solutions of therapeutic or non-therapeutic proteins can be formulated with anionic aromatic small molecule compounds as excipient compounds. Anionic aromatic excipient compounds may contain aromatic functional groups such as phenyl, benzyl, aryl, alkylbenzyl, hydroxybenzyl, phenol, hydroxyaryl, heteroaromatic groups, or condensed aromatic groups. Anionic aromatic excipient compounds may also contain anionic functional groups such as carboxylates, oxides, phenoxides, sulfonates, sulfates, phosphonates, phosphates or sulfides. Although anionic aromatic excipients may be described as acids, sodium salts or the like, it is understood that the excipients may be used in various salt forms. Without wishing to be bound by theory, it is believed that anionic aromatic excipient compounds are bulky, hindered molecules capable of binding with the cationic segments of the protein, and thus they shield these segments of the protein, thereby increasing the viscosity of protein-containing formulations. It can reduce interactions between protein molecules that provide

In embodiments, examples of anionic aromatic excipient compounds include salicylic acid, aminosalicylic acid, hydroxybenzoic acid, aminobenzoic acid, para-aminobenzoic acid, benzenesulfonic acid, hydroxybenzenesulfonic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, hydroquinone Sulfonic acid, sulfanilic acid, vanillic acid, vanillin, vanillin-taurine adduct, aminophenol, anthranilic acid, cinnamic acid, coumaric acid, adenosine monophosphate, indole acetic acid, potassium urate, furan dicarboxylic acid, furan-2-acrylic acid, 2 -furanpropionic acid, sodium petylpyruvate, sodium hydroxyphenylpyruvate, dihydroxybenzoic acid, trihydroxybenzoic acid, pyrogallol, benzoic acid, and compounds such as salts of the aforementioned acids. In an embodiment, the anionic aromatic excipient compound is formulated in the form of an ionized salt. In an embodiment, the anionic aromatic compound is formulated as a salt of a hindered amine, such as dimethylcyclohexylammonium hydroxybenzoate. In an embodiment, the anionic aromatic excipient compound is formulated with various counterions, such as organic cations. In an embodiment, a high concentration solution of a therapeutic or non-therapeutic protein is formulated with an anionic aromatic excipient compound and caffeine. In an embodiment, the anionic aromatic excipient compound is metabolized in the body to yield a biologically compatible byproduct.

c. Excipient Compound Category 3: Functionalized Amino Acids

High concentration solutions of therapeutic or non-therapeutic proteins may be formulated with one or more functionalized amino acids, wherein a single functionalized amino acid or an oligopeptide comprising one or more functionalized amino acids may be used as the excipient compound. In an embodiment, the functionalized amino acid compound comprises a molecule that can be hydrolyzed or metabolized to obtain an amino acid (“amino acid precursor”). In embodiments, the functionalized amino acid may contain aromatic functional groups such as phenyl, benzyl, aryl, alkylbenzyl, hydroxybenzyl, hydroxyaryl, heteroaromatic groups or fused aromatic groups. In embodiments, the functionalized amino acid compound may contain an esterified amino acid such as methyl, ethyl, propyl, butyl, benzyl, cycloalkyl, glyceryl, hydroxyethyl, hydroxypropyl, PEG and PPG esters. In an embodiment, the functionalized amino acid compound is selected from the group consisting of arginine ethyl ester, arginine methyl ester, arginine hydroxyethyl ester, and arginine hydroxypropyl ester. In an embodiment, the functionalized amino acid compound is a charged ionic compound in aqueous solution at neutral pH. For example, a single amino acid may be derivatized by forming an ester such as an acetate or benzoate, and the hydrolysis product would be acetic acid or benzoic acid, the natural material plus an amino acid. In an embodiment, the functionalized amino acid excipient compound is metabolized in the body to yield a biologically compatible byproduct.

d. Excipient Compound Category 4: Oligopeptides

High concentration solutions of therapeutic or non-therapeutic proteins can be formulated with oligopeptides as excipient compounds. In an embodiment, the oligopeptide is designed so that the structure has a charged segment and a bulky segment. In an embodiment, the oligopeptide consists of 2 to 10 peptide subunits. The oligopeptide may be a bifunctional, for example, a cationic amino acid bound to a non-polar amino acid or an anionic amino acid bound to a non-polar amino acid. In an embodiment, the oligopeptide consists of 2 to 5 peptide subunits. In an embodiment, the oligopeptide is a homopeptide, such as polyglutamic acid, polyasphaltic acid, poly-lysine, poly-arginine and poly-histidine. In an embodiment, the oligopeptide has a net cationic charge. In other embodiments, the oligopeptide is a heteropeptide, such as Trp2Lys3. In embodiments, the oligopeptide may have an alternating structure, such as an ABA repeat pattern. In embodiments, the oligopeptide may contain both anionic and cationic amino acids (eg, Arg-Glu). Without wishing to be bound by theory, an oligopeptide comprises a structure capable of binding with a protein in a manner that reduces intermolecular interactions resulting in highly viscous solutions; For example, an oligopeptide-protein bond may be a charge-charge interaction, leaving somewhat non-polar amino acids to disrupt the hydrogen bonds of the hydration layer around the protein, thereby lowering the viscosity. In some embodiments, the oligopeptide excipient is present in the composition at a concentration of about 50 mg/ml or less.

e. Excipient Compound Category 5: Short Chain Organic Acids

As used herein, the term "short chain organic acid" refers to a C2-C6 organic acid compound and its salts, esters or lactones. This category includes saturated and unsaturated carboxylic acids, hydroxy functionalized carboxylic acids, and linear, branched or cyclic carboxylic acids. In an embodiment, the acid group in the short chain organic acid is a carboxylic acid, a sulfonic acid, a phosphonic acid, or a salt thereof.

In addition to the above four excipient categories, high concentration solutions of therapeutic or non-therapeutic proteins can be formulated with short chain organic acids, e.g., acid and salt forms of sorbic acid, valeric acid, propionic acid, caproic acid and ascorbic acid as excipient compounds. can be formulated. Examples of excipient compounds in this category include potassium sorbate, taurine, calcium propionate, magnesium propionate and sodium ascorbate.

f. Excipient compound category 6: low molecular weight aliphatic polyacids

High concentration solutions of therapeutic or non-therapeutic pegylated proteins may be formulated with certain excipient compounds that can further lower the viscosity of the solution, wherein such excipient compounds are low molecular weight aliphatic polyacids. As used herein, the term “low molecular weight aliphatic polyacid” refers to an organic aliphatic polyacid having a molecular weight of less than about 1500 and having at least two acidic groups, wherein the acidic group is understood to be a proton-donating moiety. Non-limiting examples of acidic groups include carboxylate, phosphonate, phosphate, sulfonate, sulfate, nitrate and nitrite groups. Acidic groups for low molecular weight aliphatic polyacids may be in the form of anionic salts such as carboxylates, phosphonates, phosphates, sulfonates, sulfates, nitrates and nitrites; Their counterions may be sodium, potassium, lithium and ammonium. Specific examples of low molecular weight aliphatic polyacids useful for interacting with pegylated proteins as described herein include maleic acid, tartaric acid, glutaric acid, malonic acid, citric acid, ethylenediaminetetraacetic acid (EDTA), asphatic acid, glutamic acid, alendronic acid, etidronic acid and salts thereof. Additional examples of low molecular weight aliphatic polyacids in the form of anionic salts include phosphate (PO ₄ ^3- ), hydrogen phosphate (HPO ₄ ^{3 -} ), dihydrogen phosphate (H ₂ PO ₄ ^- ), sulfate (SO ₄ ^2- ), Bisulfate (HSO ₄ ^- ), pyrophosphate (P ₂ O ₇ ^{4 -} ), carbonate (CO ₃ ^2- ) and bicarbonate (HCO ₃ ^- ). The counterion for the anionic salt may be a Na, Li, K or ammonium ion. These excipients may also be used in combination with excipients. As used herein, a low molecular weight aliphatic polyacid can also be an alpha hydroxy acid, wherein there is a hydroxyl group adjacent to a first acidic group such as glycolic acid, lactic acid and gluconic acid and salts thereof. In an embodiment, the low molecular weight aliphatic polyacid is an oligomeric form having more than two acidic groups, such as polyacrylic acid, polyphosphate, polypeptide and salts thereof. In some embodiments, the low molecular weight aliphatic polyacid excipient is present in the composition at a concentration of about 50 mg/ml or less.

5. Protein/Excipient Solution: Characteristics and Process

In certain embodiments, solutions of therapeutic or non-therapeutic proteins are formulated with excipient compounds identified above, such as hindered amines, anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids, to mediate protein diffusion interactions. resulting in improved protein-protein interaction characteristics as measured by the variable (kD), or the second virial coefficient (B22). As used herein, "improvement" in protein-protein interaction characteristics achieved by a formulation employing the excipient compounds identified above means a reduction in protein-protein interaction. These measurements of kD and B22 can be made using standard techniques in the industry and can be indicative of improved solution properties or stability of the protein in solution. For example, a highly negative kD value may indicate that the protein has a strong attraction, which can lead to aggregation, instability, and flow problems. When formulated in the presence of certain of the above-identified excipient compounds, the same protein may have a less negative kD value or a kD value near or above zero.

In an embodiment, certain of the above-described excipient compounds, such as hindered amines, anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids and/or low molecular weight aliphatic polyacids, are subjected to heat transfer, gas transfer, centrifugation, chromatography, membrane Protein-related processes using separation, centrifugal concentration, tangential flow filtration, radial flow filtration, axial flow filtration, lyophilization and filtration by gel electrophoresis, injection, transfer, pumping, mixing, heating or cooling, such as It is used to improve the preparation, processing, sterile filtration, purification and analysis of protein-containing solutions. These processes and processing methods may have improved efficiency due to lower viscosity, improved solubility, or improved stability of the protein in solution during the manufacturing, processing, purification and analysis steps. Additionally, equipment-related processes, such as cleaning, sterilization and maintenance of protein processing equipment, would be facilitated by the use of the excipients identified above due to reduced fouling of the protein, reduced denaturation, lower viscosity and improved stability. can

A high concentration solution of a therapeutic protein formulated with the excipient compound described above can be administered to a patient using a prefilled syringe.

Example

matter:

소 감마 글로불린(BGG), 99% 초과 순도, 시그마 알드리치(Sigma Aldrich)

Bovine Gamma Globulin (BGG), >99% Purity, Sigma Aldrich

히스티딘, 시그마 알드리치

Histidine, Sigma Aldrich

이하의 실시예에 기재하는 다른 물질은 달리 구체화되지 않는 한 시그마 알드리치사로부터의 것이었다.

Other materials described in the examples below were from Sigma Aldrich unless otherwise specified.

Example 1: excipient Preparation of Formulations Containing Compounds and Test Proteins

Formulations were prepared using an excipient compound and a test protein, wherein the test protein is intended to simulate either a therapeutic protein used in a therapeutic formulation or a non-therapeutic protein used in a non-therapeutic formulation. These formulations were prepared in 50 mM histidine hydrochloride with different excipient compounds for viscosity measurement in the following way. First histidine hydrochloride was prepared by dissolving 1.94 g histidine (Sigma Aldrich, St. Louis, MO) in distilled water and adjusting the pH to about 6.0 with 1M hydrochloric acid (Sigma Aldrich, St. Louis, MO), followed by volume It was diluted to a final volume of 250 ml using distilled water in the measuring flask. The excipient compound was then dissolved in 50 mM histidine HCl. A list of excipients is provided in Examples 4, 5 and 6 below. In some cases, the excipient compound was adjusted to pH 6 prior to dissolution in 50 mM histidine HCl. In this case, the excipient compound was first dissolved in deionized water at about 5% by weight and then the pH was adjusted to about 6.0 with either hydrochloric acid or sodium hydroxide. The prepared salt solution was then placed in a laboratory oven at about 150° F. (about 65° C.) to evaporate the water and isolate the solid excipient. Once the excipient solution in 50 mM histidine HCl was prepared, the test protein (bovine gamma globulin (BGG) (Sigma-Aldrich, St. Louis, MO)) was dissolved at a ratio of about 0.336 g BGG per ml of excipient solution. This resulted in a final protein concentration of about 280 mg/ml. A solution of BGG in 50 mM histidine HCl with excipients was formulated in 20 mL vials and shaken at 100 rpm overnight on an orbital shaker table. The BGG solution was then transferred to a 2 ml centrifuge tube and centrifuged at 2300 rpm for 10 min in an IEC MicroMax microcentrifuge to remove entrained air prior to viscosity measurement.

Example 2: Viscosity measurement

Viscosity measurements of formulations prepared as described in Example 1 were performed using a DV-IIT LV conical plate viscometer (Brookfield Engineering, Middleborough, MA). The viscometer was equipped with a CP-40 cone and was operated at 3 rpm and 25°C. The formulation was loaded into the viscometer in a volume of 0.5 ml, followed by incubation for 3 minutes at the given shear rate and temperature followed by a measurement collection period of 20 seconds. This was followed by two additional steps consisting of a 1 min shear incubation followed by a 20 sec measurement collection period. The three data points collected were averaged and reported as the viscosity for the sample.

Example 3: Protein concentration measurement

The protein concentration in the experimental solution was determined by measuring the absorbance of the protein solution at a wavelength of 280 nm in a UV/VIS spectrometer (Perkin Elmer Lambda 35). Initially the instrument was calibrated to zero absorbance using 50 mM histidine buffer at pH 6. Next, the protein solution was diluted 300-fold with the same histidine buffer and the absorbance was recorded at 280 nm. The final concentration of protein in solution was calculated by using the extinction coefficient value of 1.264 ml/(mg x cm).

Example 4: steric hindrance amine Formulations with excipient compounds

Formulations containing 280 mg/ml BGG were prepared as described in Example 1, and some samples contained added excipient compounds. In these tests, hydrochloride salts of dimethylcyclohexylamine (DMCHA), dicyclohexylmethylamine (DCHMA), dimethylaminopropylamine (DMAPA), triethanolamine (TEA), dimethylethanolamine (DMEA) and niacinamide was tested as an example of a hindered amine excipient compound. Also, DMCHA and hydroxybenzoate of taurine-dicyandiamide adduct were tested as examples of hindered amine excipient compounds. The viscosity of each protein solution was measured as described in Example 2, and the results are presented in Table 1 below, which shows the advantage of the added excipient compound in reducing viscosity.

exam
number added excipients excipient concentration
(mg/ml) viscosity
(cP) viscosity
decrease 4.1 doesn't exist 0 0% 4.2 DMCHA-HCl 28 50 37% 4.3 DMCHA-HCl 43 46% 4.4 DMCHA-HCl 50 45 43% 4.5 DMCHA-HCl 82 36 54% 4.6 DMCHA-HCl 123 35 56% 4.7 DMCHA-HCl 164 40 49% 4.8 DMAPA-HCl 87 57 28% 4.9 DMAPA-HCl 40 54 32% 4.10 DCHMA-HCl 51 35% 4.11 DCHMA-HCl 50 51 35% 4.14 TEA-HCl 97 51 35% 4.15 TEA-HCl 57 28% 4.16 DMEA-HCl 51 51 35% 4.17 DMEA-HCl 41% 4.20 DMCHA-Hydroxybenzoate 67 42% 4.21 DMCHA-Hydroxybenzoate 92 42 47% 4.22 Product of Example 8 26 27% 4.23 Product of Example 8 50 37% 4.24 Product of Example 8 76 38% 4.25 Product of Example 8 42% 4.26 Product of Example 8 129 41% 4.27 Product of Example 8 159 42 47% 4.28 Product of Example 8 163 42 47% 4.29 niacinamide 48 51% 4.30 N-methyl-2-pyrrolidone 30 45 43% 4.31 N-methyl-2-pyrrolidone 52 52

Example 5: Anionic Formulations with Aromatic Excipient Compounds

A formulation of 280 mg/ml BGG was prepared as described in Example 1, and some samples contained added excipient compounds. The viscosity of each solution was measured as described in Example 2, and the results are presented in Table 2 below, which shows the advantage of the added excipient compound in reducing viscosity.

exam number added excipients Excipient concentration (mg/ml) Viscosity (cP) decrease in viscosity 5.1 doesn't exist 0 0% 5.2 Sodium Aminobenzoate 43 48 5.3 Sodium Hydroxybenzoate 26 50 37% 5.4 Sodium Sulfanilate 44 38% 5.5 Sodium Sulfanilate 96 42 47% 5.6 Indole Sodium Acetate 52 27% 5.7 Indole Sodium Acetate 27 78 One% 5.8 Vanillic acid, sodium salt 25 56 29% 5.9 Vanillic acid, sodium salt 50 50 37% 5.10 Sodium Salicylate 25 57 28% 5.11 Sodium Salicylate 50 52 5.12 Adenosine monophosphate 26 41% 5.13 Adenosine monophosphate 50 66 5.14 sodium benzoate 61 23% 5.15 sodium benzoate 56 62 22%

Example 6: Oligopeptide Formulations with excipient compounds

An oligopeptide (n=5) having an N-terminus as a free amine and a C-terminus as a free acid was synthesized by NeoBioLab Inc. with greater than 95% purity. Dipeptide (n=2) was synthesized by LifeTein LLC with 95% purity. A formulation of 280 mg/ml BGG was prepared as described in Example 1, and some samples contained synthetic oligopeptides as added excipient compounds. The viscosity of each solution was measured as described in Example 2, and the results are presented in Table 3 below, which shows the advantage of the added excipient compound in reducing viscosity.

exam number added excipients excipient concentration
(mg/ml) viscosity
(cP) viscosity
decrease 6.1 doesn't exist 0 0% 6.2 ArgX5 100 55 30% 6.3 ArgX5 50 54 32% 6.4 HisX5 100 62 22% 6.5 HisX5 50 51 35% 6.6 HisX5 25 24% 6.7 Trp2Lys3 100 59 25% 6.8 Trp2Lys3 50 24% 6.9 AspX5 100 102 -29% 6.10 AspX5 50 82 -4% 6.11 Dipeptide LE (Leu-Glu) 50 6.12 Dipeptide YE (Tyr-Glu) 50 55 30% 6.13 Dipeptide RP (Arg-Pro) 50 51 35% 6.14 Dipeptide RK (Arg-Lys) 50 53 33% 6.15 Dipeptide RH (Arg-His) 50 52 6.16 Dipeptide RR (Arg-Arg) 50 57 28% 6.17 Dipeptide RE (Arg-Glu) 50 50 37% 6.18 Dipeptide LE (Leu-Glu) 100 87 -10% 6.19 Dipeptide YE (Tyr-Glu) 100 68 14% 6.20 Dipeptide RP (Arg-Pro) 100 53 33% 6.21 Dipeptide RK (Arg-Lys) 100 19% 6.22 Dipeptide RH (Arg-His) 100 6.23 Dipeptide RR (Arg-Arg) 100 62 22% 6.24 Dipeptide RE (Arg-Glu) 100 66

Example 8: Guanyl Synthesis of taurine excipients

Guanyl taurine was prepared according to the method described in US Pat. No. 2,230,965. 3.53 parts of taurine (Sigma-Aldrich, St. Louis, MO) were mixed with 1.42 parts of dicyandiamide (Sigma-Aldrich, St. Louis, MO) and ground in a mortar until a homogeneous mixture was obtained. The mixture was then placed in a flask and heated at 200° C. for 4 hours. The product was used without further purification.

Example 9: Protein formulations containing excipient compounds

Formulations were prepared using an excipient compound and a test protein, wherein the test protein is intended to simulate either a therapeutic protein used in a therapeutic formulation or a non-therapeutic protein used in a non-therapeutic formulation. These formulations were prepared in 50 mM aqueous histidine hydrochloride buffer solution with different excipient compounds for viscosity measurement in the following way. A histidine hydrochloride buffer solution was initially prepared by dissolving 1.94 g histidine (Sigma Aldrich, St. Louis, MO) in distilled water and then adjusting the pH to about 6.0 with 1M hydrochloric acid (Sigma Aldrich, St. Louis, MO), followed by , diluted to a final volume of 250 ml with distilled water in a volumetric flask. The excipient compound was then dissolved in 50 mM histidine HCl buffer solution. A list of excipient compounds is provided in Table 4. In some cases, the excipient compound was dissolved in 50 mM histidine HCl and the resulting solution pH was adjusted with a small amount of concentrated sodium hydroxide or hydrochloric acid to achieve a pH of 6 prior to dissolution of the model protein. In some cases, the excipient compound was adjusted to pH 6 prior to dissolution in 50 mM histidine HCl. In this case, the excipient compound was first dissolved in deionized water at about 5% by weight and then the pH was adjusted to about 6.0 with either hydrochloric acid or sodium hydroxide. The prepared salt solution was then placed in a laboratory oven at about 150° F. (65° C.) to evaporate the water and isolate the solid excipient. Once the excipient solution in 50 mM histidine HCl was prepared, the test protein, bovine gamma globulin (Sigma-Aldrich, St. Louis, MO) was dissolved at a ratio to achieve a final protein concentration of about 280 mg/ml. A solution of BGG in 50 mM histidine HCl with excipients was formulated in 20 mL vials and shaken at 100 rpm overnight on an orbital shaker table. The BGG solution was then transferred to a 2 ml centrifuge tube and centrifuged at 2300 rpm for 10 min in an IEC MicroMax microcentrifuge to remove entrained air prior to viscosity measurement.

Viscosity measurements of formulations prepared as described above were performed using a DV-IIT LV cone plate viscometer (Brookfield Engineering, Middleborough, Mass.). The viscometer was equipped with a CP-40 cone and was operated at 3 rpm and 25°C. The formulation was loaded into the viscometer in a volume of 0.5 ml, followed by incubation for 3 minutes at the given shear rate and temperature followed by a measurement collection period of 20 seconds. This was followed by two additional steps consisting of a 1 min shear incubation followed by a 20 sec measurement collection period. The three data points collected were averaged and reported as the viscosity for the sample. The viscosity of the solution with excipient was normalized to the viscosity of the model protein solution without excipient. Normalized viscosity is the ratio of the viscosity of the model protein solution with excipients to the viscosity of the model protein without excipients.

exam number added excipients excipient concentration
(mg/ml) Normalized Viscosity (cP) viscosity
decrease 9.1 DMCHA-HCl 120 0.44 56% 9.2 Niacinamide 50 0.51 49% 9.3 isonicotinamide 50 0.48 52% 9.4 Tyramine HCl 70 0.41 59% 9.5 Histamine HCl 50 0.41 59% 9.6 imidazole HCl 100 0.43 57% 9.7 2-Methyl-2-imidazoline HCl 0.43 57% 9.8 1-Butyl-3-methylimidazolium chloride 100 0.48 52% 9.9 Procaine HCl 50 0.53 47% 9.10 3-Aminopyridine 50 0.51 49% 9.11 2,4,6-trimethylpyridine 50 0.49 51% 9.12 3-pyridine methanol 50 0.53 47% 9.13 Nicotinamide Adenine Dinucrel Otide 20 44% 9.15 Sodium Phenylpyruvate 55 0.57 43% 9.16 2-pyrrolidinone 0.68 32% 9.17 morpholine HCl 50 0.60 9.18 Agmatine Sulfate 55 0.77 23% 9.19 1-Butyl-3-methylimidazolium iodide 0.66 9.21 L-Anserine Nitrate 50 0.79 21% 9.22 1-Hexyl-3-methylimidazolium chloride 0.89 9.23 N,N-Diethyl Nicotinamide 50 0.67 33% 9.24 Nicotinic acid, sodium salt 100 0.54 46% 9.25 biotin 20 0.69 31%

Example 10: excipient combination and preparation of a formulation containing the test protein.

A formulation was prepared using a primary excipient compound, a secondary excipient compound and a test protein, wherein the test protein simulates either a therapeutic protein used in a therapeutic formulation or a non-therapeutic protein used in a non-therapeutic formulation. It is intended to The primary excipient compound was selected from compounds having both anionic and aromatic functional groups as listed in Table 5 below. Secondary excipient compounds were selected from compounds having either a nonionic or cationic charge and either an imidazoline or benzene ring at pH 6 as listed in Table 5 below. Formulations of these excipients were prepared in 50 mM histidine hydrochloride buffer solution for viscosity measurement in the following method. First histidine hydrochloride was prepared by dissolving 1.94 g histidine (Sigma Aldrich, St. Louis, MO) in distilled water and adjusting the pH to about 6.0 with 1M hydrochloric acid (Sigma Aldrich, St. Louis, MO), followed by volume It was diluted to a final volume of 250 ml with distilled water in the measuring flask. The individual primary or secondary excipient compounds were then dissolved in 50 mM histidine HCl. The combination of primary and secondary excipients was dissolved in 50 mM histidine HCl, and the resulting solution pH was adjusted with a small amount of concentrated sodium hydroxide or hydrochloric acid to achieve pH 6 prior to dissolution of the model protein. Once the excipient solution was prepared as described above, the test protein (bovine gamma globulin (BGG) (Sigma-Aldrich, St. Louis, MO)) was added to each test at a ratio to achieve a final protein concentration of about 280 mg/ml. Dissolve in solution.BGG solution in 50mM histidine HCl with excipients is formulated in 20ml vial and shaken at 100rpm overnight on orbital shaker table.Then, BGG solution is transferred to 2ml centrifuge tube, then IEC Entrained air was removed prior to viscosity measurement by centrifugation at 2300 rpm for 10 min in a MicroMax microcentrifuge.

Viscosity measurements of formulations prepared as described above were performed using a DV-IIT LV cone plate viscometer (Brookfield Engineering, Middleborough, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25°C. The formulation was loaded into the viscometer in a volume of 0.5 ml, followed by incubation for 3 minutes at the given shear rate and temperature followed by a measurement collection period of 20 seconds. This was followed by two additional steps consisting of a 1 min shear incubation followed by a 20 sec measurement collection period. The three data points collected were averaged and reported as the viscosity for the sample. The viscosities of the solutions with excipients were normalized to the viscosities of the model protein solutions without excipients and are summarized in Table 5 below. Normalized viscosity is the ratio of the viscosity of the model protein solution with excipients to the viscosity of the model protein without excipients. This example shows that a combination of primary and secondary excipients can provide better results than a single excipient.

Example 11: Preparation of Formulations Containing Excipient Combination and Test Protein

Formulations were prepared using a primary excipient compound, a secondary excipient compound and a test protein, wherein the test protein is used to simulate a therapeutic protein used in a therapeutic formulation or a non-therapeutic protein used in a non-therapeutic formulation. it is intended to be The primary excipient compounds were selected from compounds having both anionic and aromatic functional groups as listed in Table 6 below. Secondary excipient compounds were selected from compounds having either a nonionic or cationic charge and either an imidazoline or benzene ring at pH 6 as listed in Table 6 below. Formulations of these excipients were prepared in distilled water for viscosity measurement in the following method. The combination of primary and secondary excipients was dissolved in distilled water and the resulting solution pH was adjusted with a small amount of concentrated sodium hydroxide or hydrochloric acid to achieve pH 6 prior to dissolution of the model protein. Once the excipient solution in distilled water was prepared, the test protein, (bovine gamma globulin (BGG) (Sigma-Aldrich, St. Louis, MO)) was dissolved at a ratio to achieve a final protein concentration of about 280 mg/ml. A solution of BGG in distilled water with excipients was formulated in 20 mL vials and shaken at 100 rpm overnight on an orbital shaker table. The BGG solution was then transferred to a 2 ml centrifuge tube and centrifuged at 2300 rpm for 10 min in an IEC MicroMax microcentrifuge to remove entrained air prior to viscosity measurement.

Viscosity measurements of formulations prepared as described above were performed using a DV-IIT LV cone plate viscometer (Brookfield Engineering, Middleborough, Mass.). The viscometer was equipped with a CP-40 cone and was operated at 3 rpm and 25°C. The formulation was loaded into the viscometer in a volume of 0.5 ml, followed by incubation for 3 minutes at the given shear rate and temperature followed by a measurement collection period of 20 seconds. This was followed by two additional steps consisting of a 1 min shear incubation followed by a 20 sec measurement collection period. The three data points collected were averaged and reported as the viscosity for the sample. The viscosities of the solutions with excipients were normalized to the viscosities of the model protein solutions without excipients and are summarized in Table 6 below. Normalized viscosity is the ratio of the viscosity of the model protein solution with excipients to the viscosity of the model protein without excipients. This example shows that a combination of primary and secondary excipients can provide better results than a single excipient.

Example 12: excipient Preparation of Formulations Containing Compounds and PEG

Materials: All materials were purchased from Sigma-Aldrich, St. Louis, MO. Prepared using excipient compounds and PEG, wherein PEG is intended to simulate therapeutic PEGylated proteins used in therapeutic formulations. These formulations were prepared by mixing an equal volume of the PEG solution with the excipient solution. Solutions were both prepared at pH 7.3 in Tris buffer consisting of 10 mM Tris, 135 mM NaCl, 1 mM trans-cinnamic acid.

A PEG solution was prepared by mixing 3 g of poly(ethylene oxide) with an average Mw of approximately 1,000,000 (Aldrich Cat. No. 372781) with 97 g of Tris buffer solution. The mixture was stirred overnight for complete dissolution.

An example of excipient solution preparation is as follows: an approximately 80 mg/ml solution of citric acid in Tris buffer is prepared by dissolving 0.4 g of citric acid (Aldrich Cat. No. 251275) in 5 ml of Tris buffer solution followed by a minimal amount of 10M NaOH The solution was adjusted to pH 7.3.

The PEG excipient solution was prepared by mixing 0.5 ml of the PEG solution with 0.5 ml of the excipient solution and mixed using agitation for a few seconds. Control samples were prepared by mixing 0.5 ml of PEG solution with 0.5 ml of Tris buffer solution.

Example 13: excipient Determination of Viscosity of Formulations Containing Compound and PEG

Viscosity measurements of the prepared formulations were performed using a DV-IIT LV conical plate viscometer (Brookfield Engineering, Middleborough, Mass.). The viscometer was equipped with a CP-40 cone and was operated at 3 rpm and 25°C. The formulation was loaded into the viscometer in a volume of 0.5 ml, followed by incubation for 3 minutes at the given shear rate and temperature followed by a measurement collection period of 20 seconds. This was followed by two additional steps consisting of a 1 min shear incubation followed by a 20 sec measurement collection period. The three data points collected were averaged and reported as the viscosity for the sample.

The results provided in Table 7 show the effect of the added excipient compound in reducing the viscosity.

exam number excipient Excipient concentration (mg/ml) Viscosity (cP) decrease in viscosity 13.1 doesn't exist 0 104.8 0% 13.2 citrate Na salt 40 56.8 44% 13.3 citrate Na salt 20 73.3 28% 13.4 glycerol phosphate 40 71.7 30% 13.5 glycerol phosphate 20 83.9 18% 13.6 ethylene diamine 40 84.7 17% 13.7 ethylene diamine 20 83.9 15% 13.8 EDTA/K salt 40 67.1 36% 13.9 EDTA/K salt 20 76.9 27% 13.10 EDTA/Na salt 40 68.1 35% 13.11 EDTA/Na salt 20 77.4 26% 13.12 D-gluconic acid/K salt 40 80.32 23% 13.13 D-gluconic acid/K salt 20 88.4 13.14 D-gluconic acid/Na salt 40 81.24 23% 13.15 D-gluconic acid/Na salt 20 86.6 17% 13.16 Lactic acid/K salt 40 80.42 23% 13.17 Lactic acid/K salt 85.1 19% 13.18 Lactic acid/Na salt 40 86.55 17% 13.19 Lactic acid/Na salt 20 87.2 17% 13.20 Etidronic acid/K salt 24 71.91 31% 13.21 Etidronic acid/K salt 12 80.5 23% 13.22 Etidronic acid/Na salt 24 71.6 32% 13.23 Etidronic acid/Na salt 12 79.4 24%

Example 14: BSA 1 per molecule PEG print Genie pegylated Preparation of BSA

To a beaker was added 200 ml of phosphate buffered saline (Aldrich Cat. No. P4417) and 4 g of BSA (Aldrich Cat. No. A7906) and mixed with a magnetic bar. Next, 400 mg of methoxy polyethylene glycol maleimide, MW=5,000 (Aldrich Cat. No. 63187) was added. The reaction mixture was stirred overnight at room temperature. The next day, the reaction was stopped by adding 20 drops of 0.1M HCl. The reaction product was characterized by SDS-Page and SEC clearly showing pegylated BSA. The reaction mixture was placed in an Amicon centrifuge tube with a molecular weight cutoff (MWCO) of 30,000 and concentrated to a few milliliters. The samples were then diluted 20-fold with histidine buffer (50 mM, pH approx. 6) and concentrated until a highly viscous fluid was obtained. The final concentration of the protein solution was obtained by measuring the absorbance at 280 nm and using an extinction coefficient of 0.6678 for BSA. The results showed that the final concentration of BSA in the solution was 342 mg/ml.

Example 15: BSA multiple per molecule PEG print Genie pegylated Preparation of BSA

A 5 mg/ml solution of BSA (Aldrich A7906) in phosphate buffer (25 mM pH 7.2) was prepared by mixing 0.5 g of BSA with 100 ml of buffer. Next, 1 g of methoxy PEG propionaldehyde Mw=20,000 (JenKem Technology, Plano, 75024, Tex.) was added followed by 0.12 g of sodium cyanide borate hydride (Aldrich 156159). The reaction was allowed to proceed overnight at room temperature. The next day the reaction mixture was diluted 13-fold with Tris buffer (10 mM Tris, 135 mM NaCl, pH=7.3) and concentrated using an Amicon centrifuge tube MWCO 30,000 until approximately 150 mg/ml was reached.

Example 16: lysozyme multiple per molecule PEG print Genie pegylated lysozyme manufacture of

A 5 mg/ml solution of lysozyme (Aldrich L6876) in phosphate buffer (25 mM pH 7.2) was prepared by mixing 0.5 g of lysozyme with 100 ml of buffer. Next, 1 g of methoxy PEG propionaldehyde Mw=5,000 (JenKem Technology, Plano, 75024, Tex.) was added followed by 0.12 g of sodium cyanide borate hydride (Aldrich 156159). The reaction was allowed to proceed overnight at room temperature. The next day, the reaction mixture was diluted 49-fold with phosphate buffer (25 mM, pH 7.2) and concentrated using an Amicon centrifuge tube MWCO 30,000. The final concentration of the protein solution was obtained by measuring the absorbance at 280 nm and using the extinction coefficient for lysozyme of 2.63. The final concentration in the solution was 140 mg/ml.

Example 17: BSA 1 per molecule PEG print Genie pegylated BSA's in viscosity Effects of excipients on

A pegylated BSA formulation (from Example 14 above) with excipient was prepared by adding 6 or 12 milligrams of excipient salt to 0.3 ml of pegylated BSA solution. The solutions were mixed by gentle shaking and the viscosity was measured by a RheoSense microVisc equipped with an A10 channel (100 micron depth) at a shear rate of 500 sec-1. Viscosity measurements were completed at ambient temperature.

The results provided in Table 8 show the effect of the added excipient compound in reducing the viscosity.

exam number excipient Excipient concentration (mg/ml) Viscosity (cP) decrease in viscosity 17.1 doesn't exist 0 228.6 0% 17.2 Alpha-cyclodextrin sulfate sodium salt 20 151.5 17.3 Acetic acid K 40 89.5 60%

Example 18: BSA multiple per molecule PEG print Genie pegylated BSA's in viscosity Effects of excipients on

A formulation of pegylated BSA (from Example 15 above) with sodium citrate salt as excipient was prepared by adding 8 milligrams of excipient salt to 0.2 ml of pegylated BSA solution. The solutions were mixed by gentle shaking and the viscosity was measured by a RheoSense microVisc equipped with an A10 channel (100 micron depth) at a shear rate of 500 sec-1. Viscosity measurements were completed at ambient temperature. The results provided in Table 9 show the effect of the added excipient compound in reducing viscosity.

exam number added excipients Excipient concentration (mg/ml) Viscosity (cP) decrease in viscosity 18.1 doesn't exist 0 56.8 0% citrate Na salt 40 43.5 23%

Example 19: lysozyme multiple per molecule PEG print Genie pegylated lysozyme Effect of excipients on the viscosity of

A formulation of pegylated lysozyme (from Example 16 above) with potassium acetate as an excipient was prepared by adding 6 milligrams of excipient salt to 0.3 ml of pegylated lysozyme solution. The solutions were mixed by gentle shaking and the viscosity was measured by a RheoSense microVisc equipped with an A10 channel (100 micron depth) at a shear rate of 500 sec-1. Viscosity measurements were completed at ambient temperature. The results provided in the following table show the advantage of the added excipient compound in reducing viscosity.

exam number excipient Excipient concentration (mg/ml) Viscosity (cP) decrease in viscosity 19.1 doesn't exist 0 24.6 0% 19.2 Acetic acid K 20 22.6 8%

Example 20: excipient combination protein formulations containing

Formulations were prepared using an excipient compound, or a combination of both excipient compounds and a test protein, wherein the test protein is intended to simulate a therapeutic protein used in a therapeutic formulation. These formulations were prepared in 20 mM histidine buffer with different excipient compounds for viscosity measurement in the following way. The excipient combination was dissolved in 20 mM histidine (Sigma-Aldrich, St. Louis, MO) and the resulting solution pH was adjusted with a small amount of concentrated sodium hydroxide or hydrochloric acid to achieve a pH of 6 prior to dissolution of the model protein. The excipient compounds for this example are listed in Table 11 below. Once the excipient solution was prepared, the test protein, (bovine gamma globulin or "BGG" (Sigma-Aldrich, St. Louis, MO)) was dissolved at a ratio to achieve a final protein concentration of about 280 mg/ml. A solution of BGG in excipient solution was formulated in 5 ml sterile polypropylene tubes and shaken at 80-100 rpm overnight on an orbital shaker table. The BGG solution was then transferred to a 2 ml centrifuge tube and centrifuged at 2300 rpm for about 10 minutes in an IEC MicroMax microcentrifuge to remove entrained air prior to viscosity measurement.

Viscosity measurements of formulations prepared as described above were performed using a DV-IIT LV cone plate viscometer (Brookfield Engineering, Middleborough, Mass.). The viscometer was equipped with a CP-40 cone and was operated at 3 rpm and 25°C. The formulation was loaded into the viscometer in a volume of 0.5 ml, followed by incubation for 3 minutes at the given shear rate and temperature followed by a measurement collection period of 20 seconds. This was followed by two additional steps consisting of a 1 min shear incubation followed by a 20 sec measurement collection period. The three data points collected were averaged and reported as the viscosity for the sample. The viscosity of the solution with excipient was normalized to the viscosity of the model protein solution without excipient, and the results are shown in Table 11 below. Normalized viscosity is the ratio of the viscosity of the model protein solution with excipients to the viscosity of the model protein without excipients.

Example 21: viscosity and a protein formulation containing an excipient to reduce injection pain.

Formulations were prepared using an excipient compound, a second excipient compound and a test protein, wherein the test protein is intended to simulate the therapeutic protein used in the therapeutic formulation. The first excipient compound, Excipient A, was selected from the group of compounds having local anesthetic properties. The first excipient, Excipient A, and the second excipient, Excipient B, are listed in Table 12. These formulations were prepared in 20 mM histidine buffer using excipient A and excipient B in the following manner, and their viscosity could be measured. The amounts of excipients disclosed in Table 12 were dissolved in 20 mM histidine (Sigma-Aldrich, St. Louis, Mo.) and the resulting solution pH was adjusted with a small amount of concentrated sodium hydroxide or hydrochloric acid to achieve a pH of 6 prior to dissolution of the model protein. . Once the excipient solution was prepared, the test protein (bovine gamma globulin ("BGG") (Sigma-Aldrich, St. Louis, MO)) was dissolved in the excipient solution at a ratio to achieve a final protein concentration of about 280 mg/ml. . A solution of BGG in excipient solution was formulated in 5 ml sterile polypropylene tubes and shaken at 80-100 rpm overnight on an orbital shaker table. The BGG-excipient solution was then transferred to a 2 ml centrifuge tube and centrifuged at 2300 rpm for about 10 minutes in an IEC MicroMax microcentrifuge to remove entrained air prior to viscosity measurement.

Viscosity measurements of formulations prepared as described above were performed using a DV-IIT LV cone plate viscometer (Brookfield Engineering, Middleborough, Mass.). The viscometer was equipped with a CP-40 cone and was operated at 3 rpm and 25°C. The formulation was loaded into the viscometer in a volume of 0.5 ml, followed by incubation for 3 minutes at the given shear rate and temperature followed by a measurement collection period of 20 seconds. This was followed by two additional steps consisting of a 1 min shear incubation followed by a 20 sec measurement collection period. The three data points collected were averaged and reported as the viscosity for the sample. The viscosity of the solution with excipient was normalized to the viscosity of the model protein solution without excipient, and the results are shown in Table 12 below. Normalized viscosity is the ratio of the viscosity of the model protein solution with excipients to the viscosity of the model protein without excipients.

Example 22: Formulations containing excipient compounds and PEG

Prepared using an excipient compound and PEG, wherein the PEG is intended to simulate therapeutic PEGylated proteins used in therapeutic formulations, the excipient compounds are provided in amounts as listed in Table 13. These formulations were prepared by mixing equal volumes of the PEG solution with the excipient solution. Both solutions were prepared in deionized water.

A PEG solution was prepared by mixing 16.5 g of poly(ethylene oxide) with an average Mw of approximately 100,000 (Aldrich Cat. No. 181986) with 83.5 g of deionized water. The mixture was stirred overnight for complete dissolution.

Excipient solutions were prepared by this general method and as detailed in Table 13 below: an approximately 20 mg/ml solution of potassium triphosphate (Aldrich Cat. No. P5629) in deionized water 0.05 in 5 ml deionized water. It was prepared by dissolving g of potassium phosphate. The PEG excipient solution was prepared by mixing 0.5 ml of the PEG solution with 0.5 ml of the excipient solution and mixed using agitation for a few seconds. Control samples were prepared by mixing 0.5 ml of PEG solution with 0.5 ml of deionized water. After measuring the viscosity, the results are reported in Table 13 below.

exam number excipient excipient concentration
(mg/ml) viscosity
(cP) viscosity
decrease(%) 22.1 doesn't exist 0 79.7 0 22.2 citrate Na salt 10 74.9 6.0 22.3 potassium phosphate 10 72.3 9.3 22.4 Citrate Na Salt/Potassium Phosphate 10/10 69.1 13.3 22.5 sodium sulfate 10 75.1 5.8 22.6 Citric acid sodium salt/sodium sulfate 10/10 70.4 11.7

Example 23: Improved processing of protein solutions with excipients

Two BGG solutions were prepared by mixing 0.25 g of solid BGG (Aldrich cat # G5009) with 4 ml of buffer solution. For Sample A: The buffer solution was 20 mM histidine buffer (pH=6.0). For sample B: The buffer solution was 20 mM histidine buffer (pH=6) containing 15 mg/ml caffeine. Dissolution of solid BGG was performed by placing the sample on an orbital shaker set at 100 rpm. Buffer samples containing caffeine excipients were observed for faster dissolution of the protein. Complete dissolution of BGG was achieved within 15 minutes for the samples with caffeine excipient (Sample B). For the caffeine-free sample (Sample A), dissolution required 35 min.

The samples were then placed in two separate Amicon Ultra 4 centrifugal filter units with a 30,000 molecular weight cutoff, and the samples were centrifuged at 2,500 rpm at 10 minute intervals. The filtrate volume recovered after each 10-minute centrifugation run was reported. The results in Table 14 show a faster recovery of the filtrate for Sample B. Additionally, sample B remained concentrated by all further runs, but sample A reached its maximum concentration point and further centrifugation did not result in further sample concentration.

Centrifugation time (min) Collected Sample A filtrate (ml) Collected Sample B filtrate (ml) 10 0.28 0.28 20 0.61 30 0.78 0.88 40 0.99 1.09 50 1.27 1.42 1.51 1.71 70 1.64 1.99 1.79 2.29 90 1.79 2.39 100 1.79 2.49

Example 24: Protein formulations containing multiple excipients

This example shows how the combination of caffeine and arginine as an excipient has a beneficial effect in reducing the viscosity of a BGG solution. Four BGG solutions were prepared by mixing 0.18 g of solid BGG (Aldrich Cat# G5009) with 0.5 ml of 20 mM histidine buffer at pH 6. Each buffer solution contained a different excipient or combination of excipients as listed in the table below. The viscosity of the solution was measured as described in the previous example. The results show that the hindered amine excipient, caffeine, can be combined with known excipients such as arginine, and that the combination has better viscosity reduction than the individual excipients alone.

Arginine was added to a 280 mg/ml solution of BGG in histidine buffer at pH 6. At levels above 50 mg/ml, adding more arginine did not further reduce the viscosity as shown in Table 16.

Added arginine (mg/ml) Viscosity (cP) Viscosity reduction (%) 0 79.0 0% 53 40.9 48% 46.1 42% 105 47.8 132 49.0 38% 158 48.0 174 50.3 36% 211 51.4 35%

Caffeine was added to a 280 mg/ml solution of BGG in histidine buffer at pH 6. At levels above 10 mg/ml, adding more caffeine did not further reduce the viscosity as shown in Table 17.

Caffeine added (mg/ml) Viscosity (cP) Viscosity reduction (%) 0 0% 10 31% 15 62 23% 22 50 45%

equality

While specific embodiments of the invention have been disclosed herein, the specification is illustrative and not restrictive. While the present invention has been particularly shown and described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and description may be made therein without departing from the scope of the invention encompassed by the appended claims. . Many modifications of the invention will become apparent to those skilled in the art upon review of this specification. Unless otherwise indicated, all numbers expressing reaction conditions, amounts of ingredients, etc. in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters presented herein are approximations which may vary depending on the desired properties sought to be obtained by the present invention.

Claims (58)

A liquid formulation comprising:
a therapeutic protein and caffeine;
the caffeine is added in a viscosity-reducing amount,
(i) the viscosity-reducing amount is at least 30% less than the viscosity of the control formulation and reduces the viscosity of the formulation to a viscosity that is less than 100 cP, and (ii) the liquid formulation contains at least 100 mg/ml of the therapeutic protein. (iii) said viscosity-reducing amount of said caffeine is greater than 0 mg/ml and not more than 22 mg/ml;
the control formulation does not contain the caffeine but is the same as the liquid formulation on a dry weight basis,
wherein the therapeutic protein is an antibody. delete The liquid formulation of claim 1 , wherein the formulation is a pharmaceutical composition. delete The liquid formulation of claim 1 , wherein the viscosity of the formulation is at least 50% less than the viscosity of the control formulation. delete delete delete The liquid formulation of claim 1 , wherein the viscosity is less than 50 cP. delete delete delete delete The liquid formulation of claim 1 , wherein the formulation contains at least 200 mg/ml of the protein. 15. The liquid formulation of claim 14, wherein the formulation contains at least 300 mg/ml of the protein. delete delete The liquid formulation according to claim 1, wherein the viscosity-reducing amount is greater than 0 mg/ml and less than or equal to 15 mg/ml. delete The liquid formulation of claim 1 , wherein the caffeine is combined with a second excipient compound. 21. The liquid of claim 20, wherein the second excipient compound is selected from the group consisting of theophylline, tyramine, procaine, lidocaine, imidazole, aspartame, saccharin, acesulfame potassium, niacinamide and isonicotinamide. form. The liquid formulation of claim 1 , further comprising an additional agent selected from the group consisting of arginine and buffers. delete delete delete delete delete A method of improving protein processing, comprising:
providing the liquid formulation of claim 1, and
A method for improving protein processing, comprising using said liquid formulation in a processing method. 29. The method of claim 28, wherein the processing method is selected from the group consisting of filtration, pumping, mixing, centrifugation, membrane separation, lyophilization and chromatography. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete